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Objective 1. Generation of wild type and mutant avian influenza viruses using reverse genetics. We will use the experimental approach of generating recombinant avian influenza viruses in which the hemagglutinin gene from a moderately pathogenic (H5N3) AI virus will be transferred to a non-pathogenic virus (H5N2) and vice versa. In addition sequences with potential pathogenic potential will be removed from H5N3 and introduced into H5N2 to determine their role in pathogenesis. <p>Objective 2. In vivo characterization of AI mutant viruses. We will test the working hypothesis that amino acids differences at the cleavage site of the hemagglutinin gene of H5N3 and H5N2 are responsible for differences in pathogenicity between these two viruses. We will test this hypothesis by inoculating chickens with wild type and mutant AI viruses generated by reverse genetics.
NON-TECHNICAL SUMMARY: Avian influenza (AI) is a major respiratory disease of poultry that has the potential to cause catastrophic losses to the commercial poultry industry worldwide. The overall objective of this application is to investigate the role of the hemagglutinin of moderately pathogenic (H5N3) and non-pathogenic (H5N2) AI virus on pathogenesis in chickens.
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APPROACH: Objective 1. Generation of wild type and mutant avian influenza viruses using reverse genetics. The objective of this section is to generate wild type and mutant AI viruses in order to determine the role of the hemagglutinin gene in the pathogenesis of H5N3 virus. Generation of mutant hemagglutinin clones. The cleavage site of H5 from H5N2 and H5N3 will be mutated to contain one extra and one less basic amino acid, respectively, using standard site directed mutagenesis techniques. The presence of the desired mutations will be confirmed by DNA sequencing of these constructs. From the proposed studies we expect to identify the effect of basic amino acids in the cleavage site of the hemagglutinin gene in pathogenesis. Generation of recombinant viruses. Recombinant rH5N3, rH5N2, rH5N3-HA, rH5N2-HA, rH5N3-DHA and rH5N2+HA will be generated by transfecting the wild type and/or mutant plasmids containing the 8 genes of AI virus into 293T-CEF co-cultures. Recovered viruses will be collected and used to inoculate 10-day-old embryonated chicken eggs by the allantoic route. The titer of the viruses generated will be determined in embryonated chicken eggs and calculated as EID50/ml. <p>Objective 2. In vivo characterization of AI mutant viruses Pathogenesis of wild type and mutant viruses. Wild type and mutant AI viruses will be inoculated into 1 day and 4-week-old chickens to evaluate specific biological properties such as replication, shedding, and pathogenesis. Pathogenesis studies in 1 day-old chickens. After hatch, SPF chickens will be randomly divided into 8 groups (12 chickens per group) and inoculated intravenously (4 groups) or by the intrachoanal cleft route (4 groups) with 107 EID 50 of wild type and mutant viruses. Chicks inoculated with allantoic fluid of uninfected embryonated chicken eggs will be used as negative controls. Chickens will be observed daily for clinical signs of disease for a total of 7 days. Six chickens from each group will be euthanized at 4 and 7 days post inoculation (PI) and tissue samples taken for antibody response, histological and immunohistochemistry analysis and virus titration. Pathogenesis studies in 4 week-old chickens. After hatch, chickens will be randomly divided into 4 groups (12 chickens per group). At 4 weeks of age, each group will be inoculated with 0.2 ml/chicken containing 107 EID 50 of the wild type and mutant viruses by the intrachoanal cleft routes. Chickens inoculated with allantoic fluid of uninfected embryonated chicken eggs will be used as negative controls. Chickens will be observed daily for clinical signs of disease for a total of 7 days. Six chickens in each group will be euthanized at 4 and 7 days PI and tissue samples taken for antibody response, histological and immunohistochemistry analysis and virus titration.
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PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Using a non-pathogenic (H5N2) and a moderately (H5N3) avian influenza viruses we have generated mutant and chimera viruses in which the hemagglutinin (HA) or neuraminidase (NA) proteins have been modified. The replication properties of these viruses were evaluated in embryonated chicken eggs and no differences in virus titers were observed among the wild type and mutant viruses. The next step in this project will be to compare the pathogenicity of wild type and mutant viruses in chickens to determine the role of the HA and NA proteins, individualy and in combination, in the pathogenicity of these viruses. PARTICIPANTS: Sanjay Reddy, Associate Professor, Texas A&M University Vinayak Brahmaksatriya, Graduate Student, Texas A&M University Edu Suarez, Associate Professor, University of Puerto Rico, Ponce Noried de Jesus, Undergraduate Student, University of Puerto Rico, Ponce TARGET AUDIENCES: The target audience for this project is the scientific community, as better understanding on the pathogenesis of avian influenza viruses in chickens may lead to the development of improved vaccines and control measurements. PROJECT MODIFICATIONS: None
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IMPACT: 2007/01 TO 2007/12
New avian influenza viruses from low pathogenic strains have been generated by reverse genetics. These viruses will provide invaluable information on the role of the HA and NA proteins in the pathogenesis of these viruses.